Aromatic residues in the C-terminal domain 2 are required for Nanog to mediate LIF-independent self-renewal of mouse embryonic stem cells.

Nanog was identified by its ability to sustain the LIF-independent self-renewal of mouse embryonic stem (ES) cells and has recently been shown to play a role in reprogramming adult fibroblasts into pluripotent stem cells. However, little is known about the structural basis of these remarkable activities of Nanog. We have previously identified an unusually strong transactivator named CD2 at its C terminus. Here we demonstrate that CD2 is required for Nanog to mediate ES cell self-renewal. Furthermore, deletion and point mutation analysis revealed that CD2 relies on at least seven aromatic amino acid residues to generate its potent transactivating activity. A mutant Nanog bearing alanine substitutions for these seven residues fails to confer LIF-independent self-renewal in mouse ES cells. Substitution of CD2 by the viral transactivator VP16 gave rise to Nanog-VP16, which is 10 times more active than wild-type Nanog in ES cells. Surprisingly, the expression of Nanog-VP16 in mouse ES cells induces differentiation and is thus unable to sustain LIF-independent self-renewal for mouse ES cells. Taken together, our results demonstrate that the CD2 domain of Nanog is a unique transactivator that utilizes aromatic residues to confer specific activity absolutely required for ES self-renewal.